The Effect of Metformin and Carbohydrate-Controlled Diet on DNA Methylation and Gene Expression in the Endometrium of Women with Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is an endocrine disease associated with infertility and metabolic disorders in reproductive-aged women. In this study, we evaluated the expression of eight genes related to endometrial function and their DNA methylation levels in the endometrium of PCOS patients and women without the disease (control group). In addition, eight of the PCOS patients underwent intervention with metformin (1500 mg/day) and a carbohydrate-controlled diet (type and quantity) for three months. Clinical and metabolic parameters were determined, and RT-qPCR and MeDIP-qPCR were used to evaluate gene expression and DNA methylation levels, respectively. Decreased expression levels of HOXA10, GAB1, and SLC2A4 genes and increased DNA methylation levels of the HOXA10 promoter were found in the endometrium of PCOS patients compared to controls. After metformin and nutritional intervention, some metabolic and clinical variables improved in PCOS patients. This intervention was associated with increased expression of HOXA10, ESR1, GAB1, and SLC2A4 genes and reduced DNA methylation levels of the HOXA10 promoter in the endometrium of PCOS women. Our preliminary findings suggest that metformin and a carbohydrate-controlled diet improve endometrial function in PCOS patients, partly by modulating DNA methylation of the HOXA10 gene promoter and the expression of genes implicated in endometrial receptivity and insulin signaling.


Introduction
Polycystic ovary syndrome (PCOS) is an endocrinopathy that affects up to 20% of reproductive-aged women worldwide. PCOS is characterized by hyperandrogenism, polycystic ovaries morphology features, ovulatory dysfunction, and metabolic disorders [1,2]. Clinical features include oligo or anovulation, menstrual disorders, hirsutism, acne, obesity, dysfunction and infertility by improving hormonal parameters and IR, restoring ovulation and periodicity of the menstrual cycles [2,18,33].
Some studies have reported increased mRNA levels of some genes involved in insulin signaling and hyperplasia in the endometrium of PCOS patients after metformin treatment and lifestyle changes [19,[34][35][36], but its epigenetic effects are unidentified. Only in endometrial cancer is the precedent that metformin displays anti-tumoral properties, modifying DNA methylation and gene expression by modulating the activity of S-adenosylhomocysteine hydrolase [37,38]. However, the metformin effect on gene expression and epigenetic regulation in the endometrium of PCOS patients is widely unknown.
Particularly, the capacity of intervention with metformin and diet to modulate epigenetic and gene expression changes related to endometrial functionality in PCOS is still unexplored. Therefore, this study aimed to analyze the effect of metformin treatment and a carbohydrate-controlled diet (type and quantity) on DNA methylation and expression of selected genes related to endometrial function in endometrial tissue from patients with PCOS. We first evaluated the expression and DNA methylation levels of HOXA10, PAX6, ESR1, ESR2, IGFBP1, GAB1, SLC2A4, and IRS1 genes, as well as global DNA methylation levels in the endometrium of PCOS patients and women without the disease (control group). Then, eight of the PCOS patients underwent intervention with metformin (1500 mg/day) and a carbohydrate-controlled diet (type and quantity) for three months. We analyzed clinical variables, gene expression, and DNA methylation levels before and after the intervention in these women. Our preliminary findings suggest that metformin and a carbohydrate-controlled diet improve endometrial function in PCOS patients, partly by modulating DNA methylation of the HOXA10 gene promoter and the expression of genes implicated in endometrial receptivity and insulin signaling.

Demographic and Clinical Characteristics of Participants
All the women with PCOS recruited in the present study had polycystic ovaries (as revealed on ultrasound), oligomenorrhea, and infertility. The analysis of several clinical and biochemical variables in the early proliferative phase of the menstrual cycle revealed that PCOS patients showed higher serum levels of androstenedione, estradiol, LH/FSH ratio, and testosterone, as well as free androgen index (FAI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index than control women (Table 1). The expression of genes associated with the endometrial function (HOXA10, PAX6, ESR1, ESR2, and IGFBP1) and insulin signaling (GAB1, SLC2A4, and IRS1) was evaluated in the mid-proliferative endometrium of PCOS patients and women without the disease ( Figure 1). Results showed that gene expression of HOXA10, GAB1, and SLC2A4 was significantly lower in the endometrium of PCOS women than in controls (Figure 1A,F,G). On the other hand, the gene expression of PAX6, ESR1, ESR2, IGFBP1, and IRS1 was not statistically different between both study groups ( Figure 1).

HOXA10, GAB1, and SLC2A4 Genes Are Differentially Expressed in the Endometrium of PCOS Patients and Women without the Disease
The expression of genes associated with the endometrial function (HOXA10, PAX6, ESR1, ESR2, and IGFBP1) and insulin signaling (GAB1, SLC2A4, and IRS1) was evaluated in the mid-proliferative endometrium of PCOS patients and women without the disease ( Figure 1). Results showed that gene expression of HOXA10, GAB1, and SLC2A4 was significantly lower in the endometrium of PCOS women than in controls (Figure 1A, F, G). On the other hand, the gene expression of PAX6, ESR1, ESR2, IGFBP1, and IRS1 was not statistically different between both study groups ( Figure 1). Results are expressed as mean ± SE. * p ≤ 0.05, *** p ≤ 0.001.

The Promoter of the HOXA10 Gene Is Differentially Methylated in the Endometrium of PCOS Patients and Women without the Disease
To determine the possible role of DNA methylation in the gene expression changes observed between the endometrium of PCOS patients and women without the disease, we evaluated markers of global DNA methylation (promoter of the imprinted gene H19 and the repetitive region LINE-1) using methylated DNA immunoprecipitation (MeDIP). We found no differences in the levels of DNA methylation of H19 and LINE-1 between both study groups (Figure 2A,B). In addition, we analyzed the enrichment of this epigenetic mark on the promoters of the genes whose expression was previously determined. Interestingly, higher levels of DNA methylation at the HOXA10 gene promoter were observed in the endometrium of women with PCOS compared to controls ( Figure 2C). These findings are consistent with the lower expression of the HOXA10 gene detected in the endometrium of PCOS patients compared to women without the disease ( Figure 1A). We found no changes in the DNA methylation levels at the PAX6 and ESR1 gene promoters among both study groups ( Figure 2D,E). We did not detect DNA methylation enrichment at the promoter of ESR2, IGFBP1, GAB1, SLC2A4, and IRS1 genes in our experimental conditions in any of the study groups.

The Promoter of the HOXA10 Gene Is Differentially Methylated in the Endometrium of PCOS Patients and Women without the Disease
To determine the possible role of DNA methylation in the gene expression changes observed between the endometrium of PCOS patients and women without the disease, we evaluated markers of global DNA methylation (promoter of the imprinted gene H19 and the repetitive region LINE-1) using methylated DNA immunoprecipitation (MeDIP). We found no differences in the levels of DNA methylation of H19 and LINE-1 between both study groups (Figure 2A, B). In addition, we analyzed the enrichment of this epigenetic mark on the promoters of the genes whose expression was previously determined. Interestingly, higher levels of DNA methylation at the HOXA10 gene promoter were observed in the endometrium of women with PCOS compared to controls ( Figure 2C). These findings are consistent with the lower expression of the HOXA10 gene detected in the endometrium of PCOS patients compared to women without the disease ( Figure 1A). We found no changes in the DNA methylation levels at the PAX6 and ESR1 gene promoters among both study groups ( Figure 2D, E). We did not detect DNA methylation enrichment at the promoter of ESR2, IGFBP1, GAB1, SLC2A4, and IRS1 genes in our experimental conditions in any of the study groups.

Intervention with Metformin and a Carbohydrate-Controlled Diet Improves Hormonal and Metabolic Profiles in PCOS Women
Eight of the PCOS patients recruited in the present study underwent intervention with metformin and a carbohydrate-controlled diet for three months (PCOS + MET group; Material and Methods). Five of eight patients documented at least one menstrual cycle during the intervention; however, we cannot discard the possible effect of medroxyprogesterone acetate (used to induce menstruation in PCOS patients) in these findings. Moreover, we did not find significant changes in the size and number of ovarian cysts after the intervention.
We observed a significant decrease in the BMI, androstenedione, estradiol, insulin, and HOMA-IR index in PCOS patients after the metabolic intervention (Table 2). These results suggest that metformin and carbohydrate restriction benefit the metabolic and hormonal profile of PCOS women.

Intervention with Metformin and a Carbohydrate-Controlled Diet Induces the Expression of HOXA10, ESR1, GAB1, and SLC2A4 Genes in the Endometrium of PCOS Women
Results showed that the expression of HOXA10, ESR1, GAB1, and SLC2A4 genes was significantly higher in the PCOS + MET group than in the PCOS group ( Figure 3), suggesting that the pharmacologic and dietary intervention induced the expression of these genes in the endometrium of PCOS women. On the other hand, the gene expression of PAX6, ESR2, IGFBP1, and IRS1 was not statistically different between both the study groups.
during the intervention; however, we cannot discard the possible effect of medroxyprogesterone acetate (used to induce menstruation in PCOS patients) in these findings. Moreover, we did not find significant changes in the size and number of ovarian cysts after the intervention.
We observed a significant decrease in the BMI, androstenedione, estradiol, insulin, and HOMA-IR index in PCOS patients after the metabolic intervention (Table 2). These results suggest that metformin and carbohydrate restriction benefit the metabolic and hormonal profile of PCOS women.

Intervention with Metformin and a Carbohydrate-Controlled Diet Induces the Expression of HOXA10, ESR1, GAB1, and SLC2A4 Genes in the Endometrium of PCOS Women
Results showed that the expression of HOXA10, ESR1, GAB1, and SLC2A4 genes was significantly higher in the PCOS + MET group than in the PCOS group (Figure 3), suggesting that the pharmacologic and dietary intervention induced the expression of these genes in the endometrium of PCOS women. On the other hand, the gene expression of PAX6, ESR2, IGFBP1, and IRS1 was not statistically different between both the study groups.

Effect of Intervention with Metformin and a Carbohydrate-Controlled Diet on DNA Methylation in the Endometrium of PCOS Women
Endometrial samples obtained from women with PCOS subjected to metformin and dietary intervention showed no statistical differences in DNA methylation levels of LINE-1 and the promoter of H19, PAX6, and ESR1 genes compared to those obtained before the intervention. Interestingly, the levels of DNA methylation at the HOXA10 gene promoter were dramatically reduced in the PCOS group with the intervention (Figure 4). These findings are consistent with the higher expression of the HOXA10 gene detected in the endometrium of PCOS + MET women compared to the PCOS group without intervention ( Figure 3A).

ation in the Endometrium of PCOS Women
Endometrial samples obtained from women with PCOS subjected to metformin and dietary intervention showed no statistical differences in DNA methylation levels of LINE-1 and the promoter of H19, PAX6, and ESR1 genes compared to those obtained before the intervention. Interestingly, the levels of DNA methylation at the HOXA10 gene promoter were dramatically reduced in the PCOS group with the intervention (Figure 4). These findings are consistent with the higher expression of the HOXA10 gene detected in the endometrium of PCOS + MET women compared to the PCOS group without intervention ( Figure 3A).

Discussion
PCOS is one of the leading endocrine and metabolic disorders in premenopausal women, characterized by an alteration of the hypothalamic-pituitary-ovarian (HPO) axis, hyperandrogenism, anovulation, infertility, and insulin resistance, among other morbidities [7]. To date, the PCOS etiology remains unclear. It has been established that the endometrium is a reproductive tissue affected by PCOS partly by alterations in gene expression [39] that in turn are associated with epigenetic changes. Lifestyle intervention and metformin therapy exert positive metabolic and endocrine effects on women with PCOS and tissues affected by the disease, such as the endometrium [40]. In this preliminary study, we found that after intervention with metformin and a carbohydrate-controlled diet, the expression of the HOXA10 (related to endometrial function) and GAB1 and SLC2A4 genes (associated with insulin signaling) increased in the endometrium of eight women with PCOS. The expression of these genes decreased in the endometrium of PCOS patients compared to women without the disease. Remarkably, the increased expression of the HOXA10 gene in the endometrium of women undergoing this intervention was associated with decreased DNA methylation at its promoter. Nevertheless, we do not rule out the influence of the patients' clinical background on the observed endometrial epigenetic modifications [41][42][43][44].

Discussion
PCOS is one of the leading endocrine and metabolic disorders in premenopausal women, characterized by an alteration of the hypothalamic-pituitary-ovarian (HPO) axis, hyperandrogenism, anovulation, infertility, and insulin resistance, among other morbidities [7]. To date, the PCOS etiology remains unclear. It has been established that the endometrium is a reproductive tissue affected by PCOS partly by alterations in gene expression [39] that in turn are associated with epigenetic changes. Lifestyle intervention and metformin therapy exert positive metabolic and endocrine effects on women with PCOS and tissues affected by the disease, such as the endometrium [40]. In this preliminary study, we found that after intervention with metformin and a carbohydrate-controlled diet, the expression of the HOXA10 (related to endometrial function) and GAB1 and SLC2A4 genes (associated with insulin signaling) increased in the endometrium of eight women with PCOS. The expression of these genes decreased in the endometrium of PCOS patients compared to women without the disease. Remarkably, the increased expression of the HOXA10 gene in the endometrium of women undergoing this intervention was associated with decreased DNA methylation at its promoter. Nevertheless, we do not rule out the influence of the patients' clinical background on the observed endometrial epigenetic modifications [41][42][43][44].

Effect of the Intervention with Metformin and a Carbohydrate-Controlled Diet in Metabolic and Clinical Variables in PCOS Women
According to the diagnostic criteria for the patients included in the present study, we confirmed that several clinical parameters were altered in women with PCOS compared to women from the control group, such as androstenedione, estradiol, and testosterone serum levels, as well as the LH/FSH ratio, FAI, and IR-HOMA index (Table 1). In contrast to previous reports, we found no statistically significant changes in some clinical parameters such as BMI, DHEA, progesterone, prolactin, SHBG, glucose, and insulin between PCOS women and controls [1]. The differences between the results obtained in the present study and previous reports could be due to the characteristic phenotypic heterogeneity of the disease, and the small sample size analyzed in the present study. In addition, we did not divide the PCOS group according to BMI, which influences the phenotype of these patients [45][46][47]. Although we did not observe a dramatic reduction in the clinical parameters of the PCOS group subjected to the intervention with metformin and a carbohydratecontrolled diet, we found that BMI, androstenedione, estradiol, insulin, and IR-HOMA index decreased after the intervention (Table 2). Our results confirm the positive effects of metformin and medical nutrition therapy (MNT) in the clinical parameters of women with PCOS, as previously reported [48]. Of note, the main limitation of this study is that only eight patients were subjected to the intervention. Further studies are necessary to confirm these preliminary findings in larger sample sizes and specific PCOS phenotypes. Another limitation of this study is that we did not separately compare the effect of metformin and the carbohydrate-controlled diet. It has been demonstrated that a dietary intervention or metformin therapy alone is sufficient to improve metabolic and endocrine parameters in women with PCOS [49][50][51]. Therefore, future studies should evaluate the effect of both interventions separately on the clinical and molecular characteristics of women with PCOS.

Effect of the Intervention with Metformin and a Carbohydrate-Controlled Diet in the Expression of Genes Related to Endometrial Function in the Endometrium of PCOS Women
The gene expression analyses in the present study showed that HOXA10 gene expression decreased in the endometrium of PCOS women compared to controls ( Figure 1A), as previously reported [14,15]. Interestingly, we observed an increase in the expression of the HOXA10 gene in the patients subjected to metformin and dietary intervention ( Figure 3A). These results highlight the potential benefit of the intervention used in the present study on PCOS patients, since the HOXA10 gene is fundamental for the differentiation and receptivity of the endometrium [39]. The future assessment of gene expression (and DNA methylation) of endometrial receptivity markers such as CDH6 (cadherin 6), L-selectin ligands, osteopontin and its receptor αvβ3 integrin, and LIF is necessary to provide more information about the association among PCOS pathogenesis, endometrium receptivity, and the metformin and carbohydrate-controlled diet used in the present study [52][53][54][55].
In contrast to previous studies, we observed no changes in the expression of the ESR1 gene in the endometrium of women with PCOS compared to the endometrium of controls ( Figure 1C) [21,56,57]. Unexpectedly, the metformin and dietary intervention induced the expression of ESR1 in the endometrium of PCOS women ( Figure 3B). This finding should be explored in future studies, as deregulation of ESR1 expression is associated with endometrial hyperplasia and endometriosis [58][59][60][61].
We found no expression changes of PAX6, ESR2, IGFBP1, and IRS1 genes among the study groups, probably due to the limitations of the present study since these genes have been reported to be differentially expressed in the endometrium of women with PCOS compared to women without the disease [9, 22,23,62,63]. We also cannot discard the effect of medroxyprogesterone acetate on our findings, as it has been previously demonstrated that this synthetic hormone modifies gene expression in endometrial cells [64].
The results of the present study showed that GAB1 and SLC2A4 expression is decreased in the endometrium of women with PCOS compared to controls ( Figure 1F,G), as previously reported [18,19]. Remarkably, the expression of both genes was induced after the metformin and dietary intervention ( Figure 4C,D). In line with our results, previous studies have shown that SLC2A4 expression is restored by metformin therapy in the endometrium of women and animal models of PCOS [34,65,66]. To the best of our knowledge, this is the first report about the effects of metformin and a carbohydrate-controlled diet on the expression of genes related to balanced energy homeostasis, which is fundamental for the correct functioning of the endometrium [39].
Our preliminary results suggest that the intervention used in the present study may have beneficial effects on the endometrium function of PCOS women, which should be explored in future functional studies.

Effect of the Intervention with Metformin and a Carbohydrate-Controlled Diet on the DNA Methylation Levels of Genes Related to Endometrial Function in the Endometrium of PCOS Women
Previous studies have reported alterations in the content of DNA methylation in tissues affected by PCOS [24,32]. To our knowledge, no studies have reported differential DNA methylation levels in the endometrium of women with PCOS and women without the disease [31,67]. To explore DNA methylation as a possible mechanism of gene expression regulation in response to the intervention used in the present study, we analyzed the enrichment of 5mC at the promoters of the genes of interest using MeDIP-qPCR.
LINE-1 hypomethylation has been reported in peripheral blood cells and ovarian tissue of PCOS women. In contrast, hypermethylation of this genomic element has been found in the cumulus cells of these women [68][69][70]. In the present study, we did not detect changes in the levels of DNA methylation in the repetitive region LINE-1, neither in the endometrium of controls and PCOS women nor after the intervention. Moreover, we did not find differences in the enrichment of this epigenetic mark in the imprinted gene H19 between the study groups (Figure 2A,B). Our findings suggest that global DNA methylation is not altered in the endometrium of PCOS women; however, this finding should be confirmed using other techniques, such as DNA methylation sequencing.
The HOXA10 gene is hypermethylated in the endometrium of women with endometriosis and animal models of this disease, as well as in other endometrial diseases such as uterine polyps, intramural myoma, and submucosal myoma in which the DNA methylation status is negatively correlated with gene expression levels [71][72][73][74][75][76][77]. These reproductive diseases and PCOS are associated with infertility partly by an endometrial dysfunction that, in turn, may be explained by the increased DNA methylation at the HOXA10 promoter. In the present study, we observed a significant increase in the enrichment of 5mC at the promoter of the HOXA10 gene in the endometrium of women with PCOS compared to controls ( Figure 2C). This finding was associated with a decrease in the expression of HOXA10 in the endometrium of PCOS women ( Figure 1A), which has been previously reported [14,15].
Lifestyle intervention is a promising alternative for managing PCOS by modulating the DNA methylation content in tissues affected by the disease [78,79]. Metformin is one of the main pharmacological lines of treatment against PCOS. It has been recently reported that metformin compensates for the effect of dehydroepiandrosterone on the DNA methylation levels of oocytes in an experimental animal model of PCOS [80]. In the present study, we have shown for the first time that metformin and a carbohydrate-controlled diet decreased the DNA methylation levels in the HOXA10 gene promoter in the endometrium of PCOS women (Figure 4), which in turn was associated with an increase in its expression ( Figure 3A). These preliminary findings suggest that in PCOS women, the beneficial effects of metformin and MNT in the endometrial expression of the HOXA10 gene are partly mediated by DNA methylation. However, future studies should explore the mechanism involved, as it has been reported that metformin regulates DNA methylation by direct and indirect actions [37].
In contrast, we found no changes in the levels of PAX6 DNA methylation in the endometrium of PCOS women subjected to the intervention. Moreover, we did not observe changes in the content of DNA methylation in the promoter of the ESR1 gene that could explain the gene expression changes observed in our results. Further studies are required to explore the involvement of promoter mutations and other epigenetic mechanisms, such as histone post-translational modifications, in the gene expression changes found in the present study.

Inclusion and Exclusion Criteria for Patient Recruitment
This study was authorized and performed at the Instituto Nacional de Perinatología (INPer) in Mexico City (registry number 3000-20209-04-16). The recruitment of patients took place between 2018 and 2019. Fifteen patients diagnosed with PCOS and eight healthy women (control group) were recruited. For both groups, inclusion criteria were as follows: 18 to 45 years old (reproductive aged-women), BMI < 35 kg/m 2 , without prescription of hormonal treatment at least two months before participating in the study, and signing a written form of informed consent. Based on the Rotterdam Criteria, the inclusion criteria for PCOS patients comprised infertility, hyperandrogenism, oligo or anovulation, and polycystic ovary phenotype [5]. The inclusion criteria for women in the control group were regular menstrual cycles without PCOS symptoms. From the control and PCOS group, subjects with hormonal or GnRHa treatments, cancer diagnosis, radiotherapy or chemotherapy, chronic hypertension, diabetes mellitus, cardiac, hepatic, or renal diseases, hypo or hyperthyroidism, and asthma were excluded. Patients with infertility not related to PCOS were excluded from the PCOS group.

Sample Collection and Metabolic Intervention
Blood samples were obtained via vein puncture during the early proliferative phase of the menstrual cycle and stored in vacuum blood collection tubes with serum-separating gel; serum was further isolated via centrifugation at 1300× g at 4 • C for 10 min and stored at −70 • C. Endometrial tissue was collected using a Pipelle suction curette during the mid-proliferative phase of the menstrual cycle and stored in RNAprotect Tissue Reagent (cat. no. 76106, Qiagen, Valencia, CA, USA) at −20 • C. Before sample collection, all PCOS patients received 10 mg/day of medroxyprogesterone acetate for ten days (Provera, Pfizer, New York, NY, USA) to induce menstruation. This study was divided into two phases. In the first part of the study, we compared clinical characteristics, gene expression, and DNA methylation data between 15 women diagnosed with PCOS and 8 controls. Then, eight of the PCOS patients underwent a three-month pharmacologic intervention with oral metformin 1500 mg/day (Dabex XR, Merck, Darmstadt, Germany) and MNT with a clinical nutritionist to avoid significant variations in food intake among them (PCOS + MET group). A nutrition assessment was performed, and energy requirements were estimated with the Mifflin-St Jeor et al. equation [81] using current body weight. A controlled carbohydrate-restricted diet (40-45% of total energy intake), with 20-25% of energy from proteins and 30-35% from fat, was prescribed. The recommended diet focused on highquality carbohydrate foods (high fiber and/or low glycemic index) and promoted the intake of fruits, vegetables, low-fat dairy, legumes, whole grains, oily fish and food sources of monounsaturated fatty acids (avocados, canola oil, seeds), while limiting the intake of added sugars and ultraprocessed foods. Education themes included the healthy eating plate, healthy carbohydrates, basic carbohydrate counting, portion size estimation, and improving food choices to reduce ultra-processed foods (usually high in added sugars and/or high glycemic index). Samples from PCOS patients were obtained before and after three months of pharmacologic and dietary intervention.

DNA and RNA Isolation
Genomic DNA and total RNA were isolated with an AllPrep DNA/RNA Mini kit (cat. no. 80204, Qiagen, Valencia, CA, USA), following manufacturer instructions. DNA and RNA integrity was analyzed using 1% agarose gel electrophoresis.

RT-qPCR
cDNA synthesis was carried out using the SuperScript III First-Strand Synthesis SuperMix (cat. no. 18080400, Invitrogen, Carlsbad, CA, USA), as specified by the supplier. cDNA was subjected to PCR to amplify a gene fragment of HOXA10, PAX6, ESR1, ESR2, IGFBP1, GAB1, SLC2A4, and IRS1. ACTB (actin beta) was used as an internal control of constitutive expression. The sequences of the specific primers are listed in Table S1 of Supplementary Materials. SYBR Green Master Mix (cat. no. 4309155, Applied Biosystems, Foster City, CA, USA) was used as the detection method in a StepOne Plus Real-Time PCR system (cat. no. 4376357, Applied Biosystems, Foster City, CA, USA) following cycling conditions specified by the manufacturer. Relative quantification was performed with the ∆∆Ct method [84].

Methylated DNA Immunoprecipitation (MeDIP)-qPCR
MeDIP was carried out as previously described [85], with minor modifications. Shearing of 2 µg of DNA was performed in a Bioruptor Pico sonicator (cat. no. B01060010, Diagenode, Seraing, Belgium) by 30 cycles of 30 s ON and 30 s OFF at 20-60 kHz to obtain DNA fragments with a modal size of 200 bp, which was confirmed on a 1.5% w/v agarose gel stained with GelRed (cat. no. 41003, Biotium, Fremont, CA, USA). 1 µg of sonicated DNA was diluted in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0), denatured for 10 min at 100 • C, and snap-chilling on wet ice. 10% v/v of the sample was transferred to a clean tube for the input (normalization control). Diluted DNA was incubated with immunoprecipitation buffer (IP, 10 mM Na 2 HPO 4 , 10 mM NaH 2 PO 4 , 140 mM NaCl, 0.05% v/v Triton X-100) and 1 µg of anti-5-methylcytosine (5mC) antibody (cat. no. C15200081, Diagenode, Seraing, Belgium) at 4 • C overnight on a rotating wheel. Non-specific mouse IgG was used as a negative control antibody (cat. no. C15400001, Diagenode, Seraing, Belgium). The antibody-DNA complexes were incubated with BSA-blocked magnetic beads (Dynabeads Protein G, cat. no. 10003D Invitrogen, Carlsbad, CA, USA) in rotating agitation at 4 • C for 1 h and washed with IP buffer. Samples were incubated with digestion buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5% w/v SDS) and Proteinase K (cat. no. 25530049, Invitrogen, Carlsbad, CA, USA) overnight at 55 • C in agitation (300 rpm) to release the DNA from the beads. DNA purification was performed using phenol-chloroform-isoamyl alcohol 25:24:1 (cat. no. P2069, Sigma-Aldrich, Merck, Darmstadt, Germany) and diluted in TE buffer. qPCR with the immunoprecipitated DNA was performed using the SYBR Green method in the StepOne Plus Real-Time PCR system. In every MeDIP experiment, H19 and LINE-1 were used as endogenous methylated controls, and GAPDH promoter was used as an endogenous non-methylated control. Primers used are enlisted in Table S1. Relative levels of 5mC enrichment were determined using the ∆∆Ct method and normalized to IgG control.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 8.4.3 software (Graph Pad Software, San Diego, CA, USA). Experimental data are presented as the mean with standard deviation (SD) or standard error (SE) from three or more independent experiments. One-way ANOVA tests were performed, followed by the Tukey post hoc test. Paired data (PCOS patients before and after dietary and pharmacological intervention) were analyzed with the paired Student t-test. Kruskal-Wallis, Dunn's, and Wilcoxon signed rank tests were performed for data without normal distribution. Statistical differences were considered when p ≤ 0.05.

Conclusions
In the present study, we have shown that gene expression of HOXA10, GAB1, and SLC2A4 decreased in the endometrium of PCOS patients compared to women without the disease. Remarkably, the expression of these genes increased after metabolic intervention with metformin and a carbohydrate-controlled diet. Moreover, increased HOXA10 expres-sion after the intervention was associated with decreased DNA methylation levels at its promoter. Our preliminary findings suggest that this intervention improves endometrial function in PCOS women by modulating DNA methylation and gene expression of genes associated with endometrial receptivity and insulin signaling. Further investigation in larger sample sizes is warranted to elucidate the beneficial effects of metformin and dietary intervention on the endometrium of women with PCOS.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patients to publish this paper.

Data Availability Statement:
The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author.